Light is the primary stimulus for regulating circadian rhythms, seasonal cycles, and neuroendocrine response in many species including humans (Klein et al., 1991; Wehr. 1991). Further, clinical studies have demonstrated that light therapy is effective for treating selected affective disorders, sleep problems, and circadian disruptions (Wenerberg, 1993; Lam, 1998). Previously, the ocular photoreceptors which transduce light stimuli for circadian regulation and the clinical benefits of light therapy have been unknown.
Nonetheless, scientists have been deeply involved in elucidating the physiologic and functional anatomic features associated with light and vision. In fact, the underlying neuroanatomy and neurophysiology which mediate vision have been studied extensively over the past two centuries. More recently, the retinohypothalamic tract (RHT), a distinct neural pathway which mediates circadian regulation by environmental light, has been shown to project from the retina to the suprachiasmatic nuclei (SCN) in the hyphothalamus, (Moore R Y, Leon N J. A retinohypothalamic projection in the rat. J Comp Neruol 146:1-14, 1972; Moore R Y (1983). Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus. Federation Proceedings 42:2783-2789; Klein D C, Moore R Y, Reppert S M, eds. Suprachiasmatic Nucleus: The Mind's Clock. Oxford University Press, 5-456, 1991; Morin L P (1994). The circadian visual system, Brain Res Brain Res Rev 19:102-127). By this pathway, light and dark cycles are perceived through the mammalian eyes, entrain SCN neural activity and, in turn, entrain the rhythmic secretion of melatonin from the pineal gland. In virtually all species, melatonin secretion is high during the night and low during the day (Reiter, 1991; Arendt, 1998).
In addition to entertaining pineal rhythms, light exposure can acutely suppress melatonin secretion (Rollag and Niswender, 1976; Lewy et al., 1980). A well-defined multisynaptic neural pathway extends from the SCN to the pineal gland, which transmits information about light and circadian time for entraining the rhythmic production and secretion of the hormone melatonin. (Moore R Y, Lenn N J. J Comp Neurol 146:1-14, 1972; Klein D C et al., eds. Suprachiasmatic Nucleus: The Mind's Clock. 5-456, 1991; Schwartz W J, Busis N A, Hedley-Whyte E T. A discrete lesion of ventral hypothalamus and optic chiasm that disturbed the daily temperature rhythm. J Neurol 233:1-4, 1986; Arendt J. Melatonin and the pineal gland; influence on mammalian seasonal and circadian physiology. Rev Reprod 3:13-22, 1998). In addition to synchronizing pineal indolamine circadian rhythms, ocular exposure to light during the night can acutely suppress melatonin synthesis and secretion (Klein D C, Weller J L (1972) Rapid light-induced decrease in pineal serotonin N-acetyltransferase activity. Science 177:532-533; Lewy A J, Wehr T A, Goodwin F K, Newsome D A, Markey S P (1980) Light Suppresses melatonin secretion in humans. Science 210:1267-1269). Light-induced melatonin suppression as a well-defined, broadly used marker for photic input to the RHT and SCN (Klein D C, 1991; Arendt J (1998) Melatonin and the pineal gland: influence on mammalian seasonal and circadian physiology. Rev Reprod 3:13-22; Brainard G C, Rollag M D, Hanifin J P (1997) Photic regulation of melatonin in humans: ocular and neural signal transduction. J Biol Rhythms 12:537-546; Lucas R J, Foster R G (1999) Neither functional rod photoreceptors nor rod or cone outer segments are required for the photic inhibition of pineal melatonin. Endocrinology 140:1520-1524).
Previously, it has not been known what photoreceptors transduce light stimuli for circadian regulation. Studies on animals with hereditary or light-induced retinal degeneration have raised the possibility that neither the rods nor the cones used for vision participate in light-induced melatonin suppression, circadian locomotor phase-shifts, or photoperiodic responses (Lucas, 1999; Webb S M, Champney T H, Lewinski A K, Reiter R I (1985) Photoreceptor damage and eye pigmentation: influence on the sensitivity of rat pineal N-acetyltransferase activity and melatonin levels to light at night. Neuroendocrinology 40:205-209; Goto M, Ebihara S (1990) The influence of different light intensities on pineal melatonin content in the retinal degenerate C3H mouse and the normal CBA mouse. Neurosci Lett. 108:267-272; Foster R G, Provencio I, Hudson D, Fiske S, DeGrip W, Menaker M (1991) Circadian photoreception in the retinally degenerate mouse (rd/rd). J Camp Physiol [A] 169:39-50; Freedman M S, Lucas R J, Soni B, von Schantz M, Munoz M, David-Gray Z, Foster R G (1999) Regulation of mammalian circadian behavior by non-rod, non-cone, ocular photoreceptors. Science 284:502-504). Studies using rodents with retinal degeneration suggest that neither the rods nor cones used for vision participate in light-induced melatonin suppression, circadian phase-shifts, or photoperiodic responses (Pevet et al., 1984; Webb et al., 1985; Foster et al., 1991). Furthermore, enucleation of rod-less, cone-less transgenic mice abolishes light-induced circadian phase-shifts and melatonin suppression (Lucas and Foster, 1999; Freedman et al., 1999). Recently, light-induced melatonin suppression and circadian entrainment have been demonstrated in humans with complete visual blindness (Czeisler C A, Shanahan T L, Klennan E B, Martens H, Brotman D J, Emens J S, Klein T, Rizzo J F, III (1995) Suppression of melatonin secretion in some blind patients by exposure to bright light. (N Engl J Med 332:6-11) and with specific color vision deficiencies. (Ruberg, 1996). The study on humans with color vision deficiencies showed that protanopic and deuteranopic subjects who lacked functioning long wavelength-sensitive cones (red, or L cones), and middle wavelength cone photoreceptors (green, or M cones), exhibited normal light-induced melatonin suppression and entrainment of the melatonin rhythm (Ruberg F L, Skene D J, Hanifin J P, Rollag M D, English J, Arendt J, Brainard G C (1996) Melatonin regulation in humans with color vision deficiencies. (J Clin Endocrinol Metab 81:2980-2985). Thus, by themselves, neither the red nor green cone system could be the primary input for melatonin regulation, at least in humans with color vision deficiencies. Together, the results from human and animal circadian studies on different forms of visual blindness suggest that melatonin regulation by light is controlled, at least in part, by photoreceptors which differ from the photoreceptors that mediate vision.
Recent studies with various vertebrate species have identified several new molecules which may serve as circadian photopigments. These putative photopigments include both opsin-based molecules, such as vertebrate ancient (VA) opsin and melanopsin, as well as non-opsin molecules like the cryptochromes (Soni B G, Foster R G (1997) A novel and ancient vertebrate opsin. FEBS Lett 406:279-283; Provencio I, Jiang G, De Grip W J, Hayes W P, Rollag M D (1998) Melanopsin: an opsin in melanophores, brain, and eye. Proc Natl Acad Sci USA 95:340-345; Miyamoto Y, Sancar A (1998) Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals. (Proc Natl Acad Sci USA 95:6097-6102). Among these new photopigments, only melanopsin has been specifically localized to the human retina. (Provencio I, Rodriguez I R, Jiang G, Hayes W P, Moreira E F, Rollag M D (2000) A novel human opsin in the inner retina. J Neurosci 20:600-605. The molecular identification of these candidate photopigments and their localization in the retina and/or neural components of the circadian system make them well-suited to act as circadian phototransducers. Functional data confirming their direct role in circadian photoreception, however, have been lacking.
The present invention required deterring whether or not the three cone system, which supports photopic (daytime) vision, was also the primary input for pineal melatonin suppression in humans with normal, healthy eyes. The peak wavelength sensitivity of the photopic visual system is near 555 nm. (Rodieck R W (1998) The First Steps in Seeing, Sunderland, Mass.: Sinauer Associates, Inc.). If melatonin regulation were mediated primarily by the three cone photopic visual system, then 555 nm light would be the most potent wavelength for regulating melatonin secretion.
In the present invention, data show that 505 nm is approximately four times stronger than 555 nm in suppressing melatonin. These results demonstrate that the ocular photoreceptor primarily responsible for pineal melatonin regulation in humans, is not the cone system that is believed to mediate photopic vision. This present invention involved the first test of a specific photoreceptor system for melatonin regulation in humans with healthy, intact eyes.
Developing an action spectrum is a fundamental means for determining the input physiology for the circadian system. This photobiological technique has high utility for 1) defining the relative effectiveness of photons at different wavelengths for eliciting a biological response, and 2) identifying the specific photopigment involved in that response. (Lipson, 1994; Coohill, 1999). The specific aim of the present study was to characterize the wavelength sensitivity of the photoreceptor system responsible for providing circadian input to the human pineal gland by establishing an action spectrum for light-induced melatonin suppression. The experiments defined an action spectrum that fits a retinaldehyde opsin template and identified 446-477 nm as the most potent wavelength region for regulating melatonin. Univariance among the eight fluence-response curves suggest that a single photopigment is primarily responsible for melatonin suppression. These results suggest that there is a novel photopigment in the human eye which mediates circadian photoreception.
Light as a Therapeutic Stimulus
Numerous studies have shown that environmental light is the primary stimulus for regulating circadian rhythms, seasonal cycles, and neuroendocrine response in many mammalian species including humans (Klein et al., 1991; Morin, 1994; Czeisler, 1995). During the past 20 years, studies have tested the use of light for treating fall and winter depression (Seasonal Affective Disorder or SAD), nonseasonal depression, sleep disorders, menstrual dysfunction, and eating disorders. In addition, investigators are exploring the use of light for re-entraining human circadian physiology relative to the challenge of shift work or intercontinental air travel. A Congressional report estimated that there are 20 million shift workers in the United States. (US Congress, 1991). The two most common problems associated with shift work are reduced alertness on the job and reduced sleep quality after work. In addition, shift workers have increased health problems including higher risk of cardiovascular disease and gastrointestinal distress as well as cognitive and emotional problems. Chronic desynchronization of the circadian system is cited as on of the causes for these problems. (US Congress, 1991).
Light is known to be a potent stimulus for entraining and phase-shifting circadian rhythms in many species, including humans. (Czeisler et al., 1986; Klein et al., 1991). The circadian response to light is dependent on the stimulus intensity, wavelength and time of delivery. A phase-response curve (PRC) describes light-induced shifts in rhythms relative to the circadian phase when the light is given, and PRC's to light share similarities across many species.
Working from the human PRC to light, some investigators have tested strategies of light treatment to improve circadian entrainment thereby enhancing performance, alertness, and health in shift workers. Studying simulated shift work, different groups of investigators have shown that workers had accelerated circadian re-entrainment, enhanced alertness, and improved sleep quality after treatment with bright light (2,000 lux to 12,000 lux) versus dimmer light (10 lux to 150 lux).
Light Stimulation of the Circadian and Neuroendocrine Systems
Over the last two centuries, extensive research has elucidated the neuroanatomy and neurophysiology which support the sensory capacity of vision in mammals. More recently, animal studies have demonstrated a neural pathway, named the retinohypothalamic tract (RHT), which projects from the retina into the hypothalamus. (Moore and Lenn, 1972; Klein et al., 1991). Information about light is transmitted from the retina to the hypothalamic suprachiasmatic nuclei (SCN) which are fundamental circadian oscillators that regulate daily rhythms. (Klein et al., 1991). The pathways supporting vision and circadian regulation are anatomically separate, but there may be a link between these systems by a projection from the intergeniculate leaflet to the SCN, (Morin, 1994). Although the detailed neuroanatomy of the circadian system primarily has been determined with animal studies, parallel clinical and post-mortem studies suggest that humans have similar circadian neuroanatomy, (Schwartz et al., 1986).
The circadian system controls daily rhythms of sleep, wakefulness, body temperature, hormonal secretion, and other physiological parameters, (Klein et al., 1991; Morin, 1994; Lam, 1998). There is considerable evidence from studies on mammals that the circadian and neuroendocrine effects of light are mediated via photoreceptive physiology in the eye as opposed to photoreceptive physiology in the skin or some other part of the body. A study by Campbell and Murphy (1998), however, reported that a 3 hour bright light pulse of 13,000 lux delivered to the backs of the knees of human subjects systematically reset circadian rhythms of body temperature and melatonin. In contrast, two recent studies failed to elicit acute melatonin suppression with similar bright light exposure to the backs of the knees in healthy humans and an attempt to replicate Campbell and Murphey's findings failed to demonstrate a phase-shift after light exposure to the back of the. Further work is needed to determine whether or not the eyes are the exclusive sites for circadian photoreception in humans and other mammalian species. Data suggest that the eyes are the primary (if not exclusive) site for circadian and neuroendocrine phototransduction. Although light is the principal stimulus for regulating the circadian system, other external stimuli such as sound, temperature, social cues and conditioning may also influence physiological timing functions.
Light Regulation of Melatonin
A well-defined neural pathway carries photic information about light extends from the SCN to the pineal gland via a multisynaptic pathway with connections being made sequentially in the paraventricular hypothalamus, the upper thoracic intermediolateral cell column, and the superior cervical ganglion, (Moore, 1983). By way of this neuroanatomy, cycles of light and dark which are perceived through the eyes entrain SCN neural activity which, in turn, entrains the rhythmic synthesis and secretion of melatonin from the pineal gland. In virtually all species including humans, high levels of melatonin are secreted during the night and low levels are secreted during the day.
In addition to entraining the melatonin circadian rhythm, light can acutely suppress melatonin secretion. Specifically, exposure of the eyes to light during the night causes a rapid decrease in the high activity of the pineal enzyme serotonin-N-acetyltransferase and subsequent inhibition of synthesis and secretion of melatonin. The acute light-induced suppression of melatonin was first observed in rats and later in humans (Klein and Weller, 1972; Lewy et al., 1980). This response has been used as a tool by the PI (GCB) and many other investigators to help determine the ocular, neural and biochemical physiology for melatonin regulation, (Klein et al., 1991; Brainard et al., 1997). In addition, seasonal changes in photoperiod length alters the duration of the elevated melatonin production. Specifically, in a number of mammalian species including humans, the duration of increased nocturnal melatonin secretion is shorter in the summer due to shortened night time periods. In summary, many studies have shown that light stimuli are the strongest and most consistent regulators of melatonin. In addition, certain drugs can powerfully impact melatonin secretion, while other non-photic and non-pharmacologic stimuli that may modify melatonin levels include body posture and exercise.
Phototransduction and Action Spectrum Analysis
The overall aim of the present invention is the identification of the photoreceptor(s) for applications in the areas of circadian regulation, neuroendocrine regulation, and the clinical benefits of light therapy in humans. Fundamentally, all photobiological responses are mediated by specific organic molecules that absorb photons and then undergo physical-chemical changes which, in turn, lead to broader physiological changes within the organism. This photobiological process is termed phototransduction and the organic molecules which absorb light energy to initiate photobiological responses are called photopigments. Generally, these photoactive molecules do not absorb energy equally across the electromagnetic spectrum. Each photoreceptor molecule or complex has a characteristic absorption spectrum which depends on its atomic structure. An action spectrum is one of the main tools for the identifying the photopigment which initiates a photobiological response. The simplest definition of an action spectrum is the relative response of an organism to different wavelengths, (Lipson, 1994; Coohill, 1999).
Photobiologists have evolved a refined set of practices and guidelines for determining analytical action spectra which are applicable to all organisms from plants to humans, (Coohill, 1991; Lipson, 1994). Analytical action spectra are developed using two or more monochromatic light stimuli with half-peak bandwidths of 15 nm or less. Generally, these action spectra are determined by establishing a set of dose-response curves (fluence-response curves) at different wavelengths for a specific biological response. The action spectrum is then formed by plotting the reciprocal of incident photons required to produce the criterion biological response versus wavelength. This fundamental photobiological technique has high utility for 1) defining the relative effectiveness of different wavelengths for eliciting a biological response, and 2) identifying the specific photosensitive molecules involved in biological responses.
Action Spectra for Circadian Regulation in Rodents
As in other fields of photobiology, the initial attempts to define circadian and neuroendocrine responses to wavelength began with polychromatic action spectra which tested broader bandwidths of light in various rodent species (Coohill, 1991). These polychromatic action spectra were published mainly during the early 1970's through the mid 1980's and were reasonably consistent in indicating that the spectral region between 450 nm and 550 nm provides the strongest stimulation of circadian and neuroendocrine responses in rodents (for review: Brainard et al., 1999). Analytic action spectra, however, are superior to polychromatic action spectra for identifying the photopigments that mediate photobiological response.
In a landmark study, Takahashi and colleagues determined an analytic action spectrum for circadian wheel running behavior in Syrian hamsters (Takahashi et al., 1984). Their study established fluence-response functions for a set of monochromatic wavelengths and then formed an action spectrum from those fluence-response functions. Their action spectrum had a spectral peak (λmax) around 500 nm and seemed similar in shape to the absorption spectrum for rhodopsin. Although they found these data to support the hypothesis that a rhodopsin-based photopigment and rod cells in the retina mediate circadian entrainment in hamsters, they were careful to point out that the participation of a cone mechanism could not be excluded. Since then, three other analytic action spectra have been published on circadian and neuroendocrine regulation in rodents, (Bronstein et al., 1987; Provencio and Foster, 1995; Yoshimura and Ebihara, 1996). Data from these action spectra have been fitted to spectral sensitivity curves for retinal-based visual photopigments. This curve fitting is predicated on the assumption that a retinal-based molecule transduces light stimuli for circadian regulation, and allows the prediction of the shape of the photopigment absorption spectrum as well as its peak sensitivity (λmax). Across these rodent studies, the predicted max ranges from 480 nm to 511 nm and is surrounded by a broad region of high sensitivity. From these studies, different photopigments have been suggested to be responsible for circadian regulation including rhodopsin, a rhodopsin-like molecule, a middle wavelength cone photopigment, or a UV cone photopigment. Furthermore, preliminary data from other investigators working with Takahashi, showed that the action spectrum for photoperiod-dependent reproductive development response of male Siberian hamsters and light-induced phase-shifting of circadian locomotor activity has its λmax: in the range of 475 nm. The investigators interpret their unpublished action spectra to support the hypothesis that a short wavelength-sensitive photoreceptor mediates both functions. (Fred Turek, PhD, personal communication).
Circadian Regulation in Rodents with Loss of Cone and Rod Photoreceptors
It is important to note that there is considerable diversity in the cellular structure and function of the retina across mammalian species, and that in rodents the retina contains both cone and rod photoreceptors, (Rodieck, 1998). Early studies with blind mole rats and rats with destruction of retinal photoreceptors due to prolonged light exposure raised the possibility that neither the rods nor the cones used for vision participate in regulating the circadian and neuroendocrine systems, (Pevet et al., 1984; Webb et al., 1985). Despite profound loss of photoreceptors and vision, light detection for circadian and photoperiodic regulation was preserved. It remained possible, however, that a small population of surviving rods or cones could still be responsible for circadian photoreception.
Studies in mice with hereditary retinal disorders (rd/rd and rds/rds) have shown that these animals still exhibit normal light-induced melatonin suppression and circadian locomotor phase-shifts despite a nearly total loss of classical visual photoreceptors. The data support the conclusion that circadian photoreception is maintained either by 1) a very small number rod or cone cells, or 2) an unrecognized class of retinal photoreceptors, (Foster et al., 1991; Provencio et al., 1994; Yoshimura et al., 1994). Further work with rd mice suggested that middle-wavelength sensitive (M-cones) and/or S-cones may be responsible for circadian photoreception, (Provencio and Foster, 1995; Yoshimura and Ebihara, 1996). Recent studies with transgenic coneless (cl) mice which have extensive loss of M-cones and S-cones show that these mice exhibit normal sensitivity for light-induced melatonin suppression and circadian phase-shifting of locomotion. (Lucas et al., 1999; Freedman et al., 1999). Similarly, coneless, rodless mice (rd/rd cl) also appeared to exhibit normal sensitivity for light-induced melatonin suppression and phase-shifting of wheel-running behavior. These results indicate that rods, M-cones and S-cones are not required for circadian photoreception. Removal of the eyes however, abolished light-induced circadian phase-shifting, (Freedman et al., 1999). Overall the results suggest that the mouse eye contains specific photoreceptors for circadian regulation different from the visual photoreceptors. A study on normal rats, however, shows that the rod and cone photoreceptors for vision provide input to SCN neurons, (Aggelopoulos and Meissl, 2000). Thus, it is premature to rule out the visual photoreceptors from playing a role in circadian regulation in animals with normal, intact eyes.
If the rods and cones that mediate vision in rodents are not the primary photoreceptors for circadian regulation in rodents, what are the alternative candidates? Recent studies with various vertebrate species have identified several new molecules which may serve as circadian photopigments. These putative photopigments include both opsin-based molecules such as vertebrate ancient (VA) opsin (Soni and Foster, 1997), melanopsin (Provencio et al., 1998), and peropsin (Sun et al., 1997) as well as non-opsin molecules like biliverdin (Oren, 1996) and cryptochrome (Miyamoto and Sancar, 1998). Among these new photopigments, only melanopsin has been specifically localized to the human neural retina (Provencio et al., 2000) and cryptochrome has been localized to the mouse neural retina (Miyamoto and Sancar, 1998). The molecular identification of these candidate photoreceptors and their localization in the retina and/or neural components of the circadian system, make them well-suited to act as circadian phototransducers.
In summary, the present invention involves a light system for stimulating or regulating neuroendocrine, circadian, and photoneural systems in mammals based upon the discovery of peak sensitivity ranging from 425-505 nm. Also, the present invention involves a light meter system for quantifying light which stimulates mammalian circadian, photoneural, and neuroendocrine systems, wherein the light meter has at least one light metering device to match peak wavelength sensitivity of mammalian photoreceptors for mammalian circadian, photoneural, and neuroendocrine systems. Furthermore, the present invention exploits this peak wavelength sensitivity for novel light systems, novel translucent and transparent materials, and novel lamps or other light sources with or without filters. The present invention also involves the peak sensitivity as the focal point for treatment of mammals with a wide variety of disorders or deficits, including but not limited to, light responsive disorders, eating disorders, menstrual cycle disorders, non-specific alerting and performance deficit, hormone-sensitive cancers, and cardiovascular disorders.